Functional coupling of mu-receptor-Galphai-tethered proteins in AtT20 cells.
Opioid efficacy on mu-receptor may be influenced by various Gi/o-G-protein subunits interacting with intracellular face of receptor. Pertussis toxin-insensitive Galphai1 and Galphai2 subunits tethered with mu-receptor were stably transfected into AtT20 cells to (i) determine coupling of different alpha-subunits on opioid efficacy, and (ii) determine coupling to downstream effectors, for example, calcium and potassium channels. After pertussis toxin, stimulation of [35S]GTP-gamma-S incorporation persisted. Both constructs were able to couple to native calcium and potassium channels, with endomorphins 1 and 2 equally effective. However, pertussis toxin abolished opioid actions on calcium and potassium channels suggesting strong coupling to endogenous G-proteins, and that differences in coupling efficacy to Galphai1 and Galphai2 previously observed are restricted to initial step of signaling cascade.
Chieng
Billy C
BC
Pain Management Research Institute, Kolling Institute, University of Sydney, Royal North Shore Hospital, New South Wales, Australia. bchieng@med.usyd.edu.au
Lee
David J
DJ
Du
Yan P
YP
Osborne
Peregrine B
PB
Christie
Macdonald J
MJ
Massotte
Dominique
D
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D045744:Cell Line, Tumor; D003106:Colon / Q000378:metabolism; D004174:Diprenorphine / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D051379:Mice; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009842:Oligopeptides / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology*; D015221:Potassium Channels / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection / Q000379:methods
D000701:Analgesics, Opioid; D015220:Calcium Channels; D009842:Oligopeptides; D015221:Potassium Channels; D017450:Receptors, Opioid, mu; C105293:endomorphin 1; D004174:Diprenorphine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C105294:endomorphin 2; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go